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Mapping Protein Interactions of Sodium Channel Published online: January 15, 2018 Resource Mapping protein interactions of sodium channel NaV1.7 using epitope-tagged gene-targeted mice Alexandros H Kanellopoulos1,†, Jennifer Koenig1,†, Honglei Huang2, Martina Pyrski3 , Queensta Millet1, Stéphane Lolignier1,4, Toru Morohashi1, Samuel J Gossage1, Maude Jay1, John E Linley1,5, Georgios Baskozos6 , Benedikt M Kessler2, James J Cox1, Annette C Dolphin7, Frank Zufall3, John N Wood1,* & Jing Zhao1,** Abstract Introduction The voltage-gated sodium channel NaV1.7 plays a critical role in Pain is a major clinical problem. A 2012 National Health Interview pain pathways. We generated an epitope-tagged NaV1.7 mouse Survey (NHIS) in the United States revealed that 25.3 million adults that showed normal pain behaviours to identify channel-inter- (11.2%) suffered from daily (chronic) pain and 23.4 million acting proteins. Analysis of NaV1.7 complexes affinity-purified (10.3%) reported severe pain within a previous 3-month period under native conditions by mass spectrometry revealed 267 (Nahin, 2015). Many types of chronic pain are difficult to treat as proteins associated with Nav1.7 in vivo. The sodium channel b3 most available drugs have limited efficacy and can cause side (Scn3b), rather than the b1 subunit, complexes with Nav1.7, and effects. There is thus a huge unmet need for novel analgesics we demonstrate an interaction between collapsing-response medi- (Woodcock, 2009). Recent human and animal genetic studies have ator protein (Crmp2) and Nav1.7, through which the analgesic drug indicated that the voltage-gated sodium channel (VGSC) NaV1.7 lacosamide regulates Nav1.7 current density. Novel NaV1.7 protein plays a crucial role in pain signalling (Nassar et al, 2004; Cox et al, interactors including membrane-trafficking protein synaptotag- 2006; Dib-Hajj et al, 2013), highlighting NaV1.7 as a promising drug min-2 (Syt2), L-type amino acid transporter 1 (Lat1) and trans- target for development of novel analgesics (Habib et al, 2015; membrane P24-trafficking protein 10 (Tmed10) together with Emery et al, 2016). Scn3b and Crmp2 were validated by co-immunoprecipitation (Co- VGSCs consist of a large pore-forming a-subunit (~260 kDa) IP) from sensory neuron extract. Nav1.7, known to regulate opioid together with associated auxiliary b-subunits (33–36 kDa) and play receptor efficacy, interacts with the G protein-regulated inducer of a fundamental role in the initiation and propagation of action poten- neurite outgrowth (Gprin1), an opioid receptor-binding protein, tials in electrically excitable cells. Nine isoforms of a-subunits demonstrating a physical and functional link between Nav1.7 and (NaV1.1–1.9) that display distinct expression patterns, and variable opioid signalling. Further information on physiological interactions channel properties have been identified in mammals (Frank & provided with this normal epitope-tagged mouse should provide Catterall, 2003). NaV1.7, encoded by the gene SCN9A in humans, is useful insights into the many functions now associated with the selectively expressed peripherally in dorsal root ganglion (DRG), NaV1.7 channel. trigeminal ganglia and sympathetic neurons (Toledo-Aral et al, 1997; Black et al, 2012), as well as in the central nervous system Keywords NaV1.7; pain; protein–protein interactor; sensory neuron; sodium (Weiss et al, 2011; Branco et al, 2016). As a large membrane ion channel channel, NaV1.7 produces a fast-activating, fast-inactivating and Subject Categories Molecular Biology of Disease; Neuroscience slowly repriming current (Klugbauer et al, 1995), acting as a thres- DOI 10.15252/embj.201796692 | Received 5 April 2017 | Revised 30 November hold channel to contribute to the generation and propagation of 2017 | Accepted 5 December 2017 | Published online 15 January 2018 action potentials by amplifying small sub-threshold depolarisations The EMBO Journal (2018) 37: 427–445 (Rush et al, 2007). The particular electrophysiological characteris- tics of NaV1.7 suggest that it plays a key role in initiating action 1 Molecular Nociception Group, WIBR, University College London, London, UK 2 TDI Mass Spectrometry Laboratory, Target Discovery Institute, University of Oxford, Oxford, UK 3 Center for Integrative Physiology and Molecular Medicine, Saarland University, Homburg, Germany 4 Université Clermont Auvergne, Inserm U1107 Neuro-Dol, Pharmacologie Fondamentale et Clinique de la Douleur, Clermont-Ferrand, France 5 Neuroscience, IMED Biotech Unit, AstraZeneca, Cambridge, UK 6 Division of Bioscience, University College London, London, UK 7 Department of Neuroscience, Physiology and Pharmacology, University College London, London, UK *Corresponding author. Tel: +44 207 6796 954; E-mail: [email protected] **Corresponding author. Tel: +44 207 6790 959; E-mail: [email protected] † These authors contributed equally to this work ª 2018 The Authors. Published under the terms of the CC BY 4.0 license The EMBO Journal Vol 37 |No3 | 2018 427 Published online: January 15, 2018 The EMBO Journal NaV1.7-interacting proteins Alexandros H Kanellopoulos et al potentials in response to depolarisation of sensory neurons by (Fig 1B). The channel function of TAP-tagged NaV1.7 was examined noxious stimuli (Habib et al, 2015). In animal studies, our previous with electrophysiological analysis, and the result showed that all the results demonstrated that conditional NaV1.7 knockout mice have cells presented normal functional NaV1.7 currents (Fig 1C). Activa- major deficits in acute, inflammatory and neuropathic pain (Nassar tion and fast inactivation data were identical for wild-type NaV1.7 et al, 2004; Minett et al, 2012). Human genetic studies show that and TAP-tagged NaV1.7 (Fig 1D) demonstrating that the TAP-tag loss-of-function mutations in NaV1.7 lead to congenital insensitivity does not affect the channel function of NaV1.7. to pain, whereas gain-of-function mutations cause a range of painful inherited disorders (Cox et al, 2006; Dib-Hajj et al, 2013). Recent Generation of a TAP-tagged NaV1.7 mouse studies also show that NaV1.7 is involved in neurotransmitter release in both the olfactory bulb and spinal cord (Weiss et al, 2011; We used a conventional gene targeting approach to generate an Minett et al, 2012). Patients with recessive NaV1.7 mutations are epitope-tagged NaV1.7 mouse. The gene targeting vector was normal (apart from being pain-free and anosmic), suggesting that constructed using an Escherichia coli recombineering-based method selective NaV1.7-blocking drugs are therefore likely to be effective (Bence et al, 2005; Fig EV1). A TAP-tag, which contains a HAT analgesics with limited side effects (Cox et al, 2006). domain, a TEV cleavage site and 3x FLAG-tags (Fig 1A), was Over the past decade, an enormous effort has been made to inserted into the open reading frame at the 30-end prior to the stop develop selective NaV1.7 blockers. Efficient and selective NaV1.7 codon in exon 27 of NaV1.7 (NCBI Reference: NM_001290675; 0 antagonists have been developed; however, NaV1.7 antagonists Fig 2A). The final targeting vector construct containing a 5 homol- require co-administration of opioids to be fully analgesic (Minett et al, ogy arm (3.4 kb), a TAP-tag, neomycin cassette and a 30 homology 2015). NaV1.7 thus remains an important analgesic drug target and an arm (5.8 kb; Fig 2B) was transfected into the 129/Sv embryonic alternative strategy to target NaV1.7 could be to interfere with either stem (ES) cells. Twelve colonies with the expected integration (tar- intracellular trafficking of the channel to the plasma membrane, or the geting efficiency was 3.5%) were detected by Southern blot analyses downstream effects of the channel on opioid peptide expression. (Fig 2C). Germline transmission and intact TAP-tag insertion after Although some molecules, such as Crmp2, ubiquitin ligase Nedd4-2, removal of the neomycin (neo) cassette were confirmed with South- fibroblast growth factor 13 (FGF13) and PDZ domain-containing ern blot, PCR and RT–PCR, respectively (Fig 2D–F). This mouse line TAP protein Pdzd2, have been reported to associate with the regulation of is henceforth referred to as NaV1.7 . trafficking and degradation of NaV1.7, the entire protein–protein inter- TAP action network of NaV1.7 still needs to be defined (Shao et al,2009; NaV1.7 mice have normal pain behaviour Dustrude et al, 2013; Laedermann et al, 2013; Bao, 2015; Yang et al, TAP 2017). Affinity purification (AP) and targeted tandem affinity purifica- The homozygous NaV1.7 knock-in mice (KI) were healthy, tion (TAP) combined with mass spectrometry (MS) is a useful method fertile and apparently normal. Motor function of the mice was for mapping the organisation of multiprotein complexes (Angrand examined with the Rotarod test. The average time that KI animals et al, 2006; Wildburger et al, 2015). Using the AP-MS or TAP-MS stayed on the rod was similar to the WT mice (Fig 3A), suggest- method to characterise protein complexes from transgenic mice allows ing there is no deficit on motor function in the KI mice. The the identification of complexes in their native physiological environ- animal responses to low-threshold mechanical, acute noxious ment in contact with proteins that might only be specifically expressed thermal, noxious mechanical stimuli and acute peripheral in certain tissues (Fernandez et al, 2009). The principal aim of this inflammation were examined with von Frey filaments, Harg- study was to identify new protein interaction partners and networks reaves’ test, Randall–Selitto apparatus and formalin test, respec- involved in the trafficking and regulation of the sodium channel tively. The results showed that TAP-tagged KI mice had identical NaV1.7 using mass spectrometry and our recently generated epitope responses to these stimuli compared to the littermate WT control TAP (TAP)-tagged NaV1.7 knock-in mouse. Such information should throw mice (Fig 3B–E), indicating NaV1.7 mice have normal acute new light on the mechanisms through which NaV1.7 regulates a vari- pain behaviour.
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